EP2675924A2 - Tests de mutations ponctuelles par pcr en temps réel pour détecter la résistance du vih-1 aux médicaments antiviraux - Google Patents

Tests de mutations ponctuelles par pcr en temps réel pour détecter la résistance du vih-1 aux médicaments antiviraux

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Publication number
EP2675924A2
EP2675924A2 EP12747199.3A EP12747199A EP2675924A2 EP 2675924 A2 EP2675924 A2 EP 2675924A2 EP 12747199 A EP12747199 A EP 12747199A EP 2675924 A2 EP2675924 A2 EP 2675924A2
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EP
European Patent Office
Prior art keywords
seq
primer
mutation
hiv
reverse
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP12747199.3A
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German (de)
English (en)
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EP2675924A4 (fr
Inventor
Jeffrey A. Johnson
Walid M. Heneine
Jonathan T. LIPSCOMB
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US Department of Health and Human Services
US Government
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US Department of Health and Human Services
US Government
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Publication of EP2675924A2 publication Critical patent/EP2675924A2/fr
Publication of EP2675924A4 publication Critical patent/EP2675924A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • HIV pandemic now exceeds 40 million persons and its expansion is being met with an increased use of anti-HIV drugs to care for the lives of those affected. Emergence of drug resistance is expected to increase as the use of these drugs for the clinical management of HIV-1 infected persons increases worldwide.
  • Highly active antiretroviral therapy (HAART) containing a combination of three antiretroviral drugs is currently recommended and has been effective in reducing mortality and morbidity.
  • ⁇ drugs that inhibit either virion entry (e.g., T-20), nucleotide extension by viral reverse transcriptase (e.g., 3TC, d4T), reverse transcriptase enzymatic activity (e.g., nevirapine, efavirenz), or the viral protease (e.g., nelfinavir, lopinavir).
  • virion entry e.g., T-20
  • nucleotide extension by viral reverse transcriptase e.g., 3TC, d4T
  • reverse transcriptase enzymatic activity e.g., nevirapine, efavirenz
  • the viral protease e.g., nelfinavir, lopinavir.
  • Drug resistance that is conferred by mutations is frequently selected in viruses from patients failing antiretroviral therapy and is considered a major cause of treatment failure.
  • Drug resistance testing is performed through the use of phenotypic or genotypic assays. Phenotypic assays measure drug susceptibilities of patient-derived viruses and provide direct evidence of drug resistance. However, phenotypic assays are culture-based, complex, laborious, and costly. Genotypic assays are frequently used to detect mutations associated with drug resistance by sequence analysis of the viral RNA from plasma.
  • Drug resistance testing is also indicated for patients receiving HAART to manage treatment failures and to help guide the selection of new regimens with active drugs.
  • Recent data have pointed to the importance of sensitive drug resistance assays for this testing and associate low-frequency drug-resistant viruses that are not detectable by conventional sequencing with poor treatment outcomes (Mellors et al., 1 1 th CROI, 2004; Jourdain et al., JID 2004) (1).
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • the greater sensitivity of the present real-time PCR resistance assays over conventional sequencing may allow earlier detection of resistance mutations that emerge during treatment and provide advance notice of possible declines in response to therapy. Early detection will help guide clinicians in modifying drug regimens in an effort to prevent treatment failure and the emergence of high-level drug resistance.
  • Methods with greater sensitivity in detecting low levels of resistant virus, below what is capable by conventional sequence analysis, are important for improving clinical management of patients under HAART.
  • the substantially higher sensitivity, the simplicity, the high throughput capability, and the low cost of the present real-time PCR drug resistance assays are all advantages over conventional sequence analysis.
  • compositions including primers and probes which are capable of interacting with the disclosed nucleic acids, such as the nucleic acids encoding the reverse transcriptase, protease, or integrase of HIV as disclosed herein and methods of highly sensitive mutation specific detection of drug resistant HIV in biological samples.
  • an oligonucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NOST-89, 96-122, and 124-141.
  • an oligonucleotide consisting of any of the nucleotide sequences set forth in SEQ ID NOS:l-89, 96-122, and 124-141.
  • Each of the disclosed oligonucleotides is a probe or a primer.
  • mixtures of primers and mixtures of primers and probes and for use in RT-PCR and primary PCR reactions disclosed herein. Kits comprising the primers or probes are provided. Provided are methods for the specific detection of several mutations in HIV. Mutations in the reverse
  • transcriptase, protease, and integrase of HIV can be detected using the methods described herein.
  • FIG. 1 A and I B are schematic illustrations of the principle of the present assay
  • FIG. 2A shows the sensitivity of the assay for 184V
  • FIG. 2B shows the lower limit for 184V detection in clinical specimens
  • FIG. 3 shows the performance of the 184V assay on clinical specimens
  • FIG. 4 shows the ACT frequency distribution for clinical samples
  • FIG. 5 shows the ACT values for real-time PCR analysis of the 103N mutation in pre-NVP and post-NVP plasma samples
  • FIG. 6 shows the clinical and absolute detection limits of a method of detection of the HIV-1 subtype C 65 R mutation.
  • FIG. 7 shows the absolute detection limit of a method of detection of the HIV-1 integrase 148R mutation.
  • PCR-based point mutation assays In an effort to improve the detection of mutations associated with HIV-1 drug resistance, provided are PCR-based point mutation assays.
  • the present methodology allows testing for different point mutations in patient samples at an achievable sensitivity of 1 -2 log greater than conventional sequencing.
  • the invention has utility to detect the presence or absence of a drug-resistant strain of HIV.
  • the principle of the present assay is to compare the differential amplifications of a mutation-specific PCR and a total copy (common) PCR, which detects all sequences present.
  • the assay can use template generated from RT-PCR of viral RNA or from PCR of proviral DNA from infected cells (Fig. 1 ).
  • 103N and 184V Two important HIV-1 reverse transcriptase mutations that significantly compromise the success of treatment with reverse transcriptase inhibitors are 103N and 184V.
  • the 103N mutation is frequently selected in patients failing treatment with non-nucleoside RT inhibitors (e.g., nevirapine, efavirenz).
  • non-nucleoside RT inhibitors e.g., nevirapine, efavirenz
  • 184V mutation following exposure to nucleoside inhibitors lamivudine (3TC), emtricitabine (FTC), and abacavir, and it's seemingly rapid disappearance after discontinuation of therapy, makes accurate measure of rapidly decaying mutations important for surveillance and clinical management.
  • the methods disclosed herein have multiple applications including (1) resistance testing for clinical management of HIV-infected persons receiving anti- HIV drugs (for detecting emergence of resistant viruses in treated persons, and as a pre-treatment evaluation of patient baseline HIV in order to tailor the most appropriate drug combination), (2) use in blood bank screening as a nucleic acid test (NAT), due to the high sensitivity and high throughput capability of the assays, (3) the ability to measure plasma viral loads, since the assays are inherently quantitative, (4) use as a screening tool for monitoring the spread of resistant HIV, (5) use as a research tool to study the emergence and biology of drug resistance mutations, (6) detection of resistance mutations in both subtype B and non-B subtypes of HIV-1, (7) possible detection of resistance mutations in HIV-2, and (8) identification of specific panels of mutations that are designed to address each of the described uses.
  • the reagents and specific usages developed here are unique.
  • compositions including primers and probes, which are capable of interacting with the disclosed nucleic acids, such as the nucleic acids encoding the reverse transcriptase or protease of HIV as disclosed herein and in the literature.
  • an oligonucleotide comprising a nucleotide sequence as set forth in any of SEQ ID OS: l-89, 96-122, and 124-141. Also provided is an oligonucleotide consisting of any one of the nucleotide sequences set forth in SEQ ID NOS: 1-89, 96-122, and 124-141. Thus, provided is an oligonucleotide comprising the sequence selected from the group consisting of the nucleotides as set forth in the sequence listing as SEQ ID NOS: 1-89, 96-122, and 124-141.
  • Each of the disclosed oligonucleotides is a probe or a primer. Each can be used independently of the others in an amplification method or in a hybridization/probing method. One or more of the probes or primers can be used together in the
  • compositions and methods for detecting mutations are described herein.
  • a nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an intemucleoside linkage.
  • the base moiety of a nucleotide can be adenin-9-yl (A), cytosin-l-yl (C), guanin-9-yi (G), uracil-l-yl (U), and thymin-l -yl (T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • a non-limiting example of a nucleotide would be 3'-AMP (3'-adenosine
  • nucleotide includes nucleotides and nucleotide analogs, preferably groups of nucleotides comprising oligonucleotides, and refers to any compound containing a heterocyclic compound bound to a phosphorylated sugar by an N-glycosyl link or any monomer capable of complementary base pairing or any polymer capable of hybridizing to an oligonucleotide.
  • nucleotide analog refers to molecules that can be used in place of naturally occurring bases in nucleic acid synthesis and processing, preferably enzymatic as well as chemical synthesis and processing, particularly modified nucleotides capable of base pairing.
  • a nucleotide analog is a nucleotide which contains some type of modification to one of the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-mefhylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.
  • This term includes, but is not limited to, modified purines and pyrimidines, minor bases, convertible nucleosides, structural analogs of purines and pyrimidines, labeled, derivatized and modified nucleosides and nucleotides, conjugated nucleosides and nucleotides, sequence modifiers, terminus modifiers, spacer modifiers, and nucleotides with backbone modifications, including, but not limited to, ribose-modified nucleotides, phosphoramidates, phosphorothioates, phosphonamidites, methyl phosphonates, methyl phosphoramidites, methyl phosphonamidites, 5'- ⁇ -cyanoethyl phosphoramidites, methylenephosphonates, phosphorodithioates, peptide nucleic acids, achiral and neutral internucleotidic linkages and nonnucleotide bridges such as polyethylene glycol, aromatic
  • nucleotide analog is a synthetic base that does not comprise adenine, guanine, cytosine, thymidine, uracil or minor bases.
  • nucleotide and nucleoside derivatives, analogs and backbone modifications are known in the art (e.g., Picciril!i J. A. et al. (1990) Nature 343:33-37; Sanghvi et al (1993) in: Nucleosides and Nucleotides as Antitumor and Antiviral Agents, (Eds. C. K. Chu and D. C. Baker) Plenum, New York, pp. 31 1-323; Goodchild J. (1990) Bioconjugate Chemistry 1 :165-187; Beaucage et al. (1993) Tetrahedron 49: 1925- 1963).
  • Nucleotide substitutes include molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • nucleic acid based There are a variety of molecules disclosed herein that are nucleic acid based.
  • nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein.
  • oligonucleotide means a naturally occurring or synthetic polymer of nucleotides, preferably a polymer comprising at least three nucleotides and more preferably a polymer capable of hybridization. Oligonucleotides may be single- stranded, double-stranded, partially single-stranded or partially double- stranded ribonucleic or deoxyribonucleic acids, including selected nucleic acid sequences, heteroduplexes, chimeric and hybridized nucleotides and oligonucleotides conjugated to one or more nonoligonucleotide molecules.
  • nucleotides comprising a oligonucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose.
  • deoxyribonucleotides such as adenine, cytosine, guanine or thymine linked to 2'-deoxyribose
  • ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose.
  • oligonucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides.
  • nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry 34: 11363-1 1372 (1 95); Pagratis et al, Nature Biotechnol. 15:68-73 (1997), each of which is incorporated herein by reference).
  • polynucleotide is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond.
  • polynucleotide includes RNA and DNA, which can be a gene or a portion thereof, a cD A, a synthetic poly deoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid.
  • polynucleotide as used herein includes naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic molecules, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a polynucleotide of the invention can contain nucleoside or nucleotide analogs, or a backbone bond other than a phosphodiester bond.
  • the nucleotides comprising a polynucleotide are naturally occurring
  • deoxyribonucleotides such as adenine, cytosine, guanine or thymine linked to 2'- deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose.
  • a polynucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Such nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res.
  • the covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond.
  • the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tarn et al., Nucl.
  • nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the polynucleotide is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium or upon administration to a living subject, since the modified polynucleotides can be less susceptible to degradation.
  • Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.
  • Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.
  • Primers are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • oligonucleotides of SEQ ID NOS: l-89, 96-122, and 124-141 can be modified in insubstantial ways and yet retain substantially the same hybridization strength and specificity as described herein. These parameters are easily measured in assays such as those taught herein. Thus, one of skill in the art will be able to envision a number of nucleotide substitutions to the disclosed sequences, so long as they retain 80% sequence similarity with the specifically disclosed sequence.
  • Primers and probes of the invention can include sequences having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% similarity to one of SEQ ID NOS:l-89, 96-122, and 124-141 are envisioned. More specifically, primers and probes with substitutions based on known sequences of the HIV-1 protease, reverse transcriptase, or integrase are envisioned because these alternative sequences are envisioned by the person of skill in this art.
  • the primers are used to support DN A amplification reactions.
  • the primers are capable of being extended in a sequence specific manner.
  • Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer.
  • Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, R A transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred.
  • the primers are used for the DNA amplification reactions, such as PCR or direct sequencing.
  • the primers can also be extended using non- enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner.
  • the disclosed primers hybridize with the disclosed nucleic acids or region of the nucleic acids or they hybridize with the complement of the nucleic acids or complement of a region of the nucleic acids.
  • hybridizes under stringent conditions describes conditions for hybridization and washing under which nucleotide sequences having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, base pair matches to each other typically remain hybridized to each other.
  • Illustrative hybridization conditions are described in, for example but not limited to, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
  • a non-limiting example of stringent hybridization conditions is hybridization in 6x sodium chloride/sodium citrate (SSC), 0.5% SDS at about 60°C followed by one or more washes in 2xSSC, 0.5% SDS at room temperature.
  • SSC sodium chloride/sodium citrate
  • stringent hybridization conditions hybridization in 6x SSC at about 45 °C followed by one or more washes in 0.2x SSC, 0.1% SDS at 50to 65 °C.
  • Other stringent hybridization conditions will be evident to one of ordinary skill in the art based on general knowledge in the art as well as this specification.
  • nucleotide or oligonucleotide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the nucleotide is derived, or is substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a nucleotide/oligonucleotide in which the nucleotide/oligonucleotide is separated from cellular components of the cells from which it is isolated or produced.
  • a nucleotide/oligonucleotide that is substantially free of cellular material includes preparations of the nucleotide having less than about 30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry weight) of contaminating material.
  • nucleotide/oligonucleotide is produced by chemical synthesis, it is optionally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the molecule. Accordingly, such preparations of the
  • nucleotide/oligonucleotide have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the nucleotide/oligonucleotide of interest.
  • a nucleotide/oligonucleotide is isolated or purified.
  • sample is a portion of a larger source.
  • a sample is optionally a solid, gaseous, or fluidic sample.
  • a sample is illustratively an environmental or biological sample.
  • An environmental sample is illustratively, but not limited to, water, sewage, soil, or air.
  • a “biological sample” is as sample obtained from a biological organism, a tissue, cell, cell culture medium, or any medium suitable for mimicking biological conditions.
  • Non-limiting examples include, saliva, gingival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogenital secretions, synovial fluid, blood, serum, plasma, urine, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, intracellular fluid, ocular fluids, seminal fluid, mammary secretions, and vitreal fluid, and nasal secretions, throat or nasal materials.
  • target agents are contained in: CSF; serum; whole blood; throat fluid; nasopharyngeal fluid; or other respiratory fluid.
  • medium refers to any liquid or fluid sample in the presence or absence of a bacterium.
  • a medium is illustratively a solid sample that has been suspended, solubilized, or otherwise combined with fluid to form a fluidic sample.
  • Non-limiting examples include buffered saline solution, cell culture medium, acetonitrile, trifluoroacetic acid, combinations thereof, or any other fluid recognized in the art as suitable for combination with bacteria or other cells, or for dilution of a biological sample or amplification product for analysis.
  • oligonucleotides described herein include primers and probes optionally effective for cross subtype reactive PCR, as such, they are capable of detecting mutations in a variety of HIV subtypes.
  • the following primers and probes can also include additions known to those skilled in the art. Examples of such additions include, but are not limited to, molecules for linking the primer to a substrate, and the like.
  • a nucleic acid molecule of the invention can be any nucleic acid molecule of the invention.
  • detectable moiety is intended to mean any suitable label, including, but not limited to, enzymes, fluorophores, biotin, chromophores, radioisotopes, colored particles, electrochemical, chemical-modifying or chemiluminescent moieties. Examples include (i) enzymes which can catalyze color or light emitting (luminescence) reactions and (ii) fluorophores.
  • the detection of the detectable moiety can be direct provided that the detectable moiety is itself detectable, such as, for example, in the case of
  • the detection of the detectable moiety can be indirect. In the latter case, a second moiety reactable with the detectable moiety, itself being directly detectable is preferably employed.
  • the detectable moiety may be inherent to a molecular probe. Common fluorescent moieties include: fluorescein, cyanine dyes, coumarins, phycoerythrin, phycobiliproteins, dansyl chloride, Texas Red, and lanthanide complexes.
  • primers for use in RT-PCR and primary PCR reactions disclosed herein.
  • a mixture of primers comprising SEQ ID NO:l and 3 is provided.
  • This mixture can be used for the reverse transcription-PCR (RT-PCR) reaction and the primary PCR reaction for HIV. It reverse transcribes and amplifies the HIV protease region comprising positions 30 and 90 in addition to the region of the reverse transcriptase gene comprising the mutations described herein.
  • This mixture does not reverse transcribe or amplify the protease regions of interest, but is useful for the analysis of the reverse transcriptase.
  • a mixture of primers comprising SEQ ID NOS: 4 and 6 is provided. This mixture is for the RT-PCR and primary PCR reactions for HIV. It also reverse transcribes and amplifies the HIV protease region comprising positions 30 and 90 in addition to the region of the reverse transcriptase gene comprising the mutations described herein.
  • a mixture of primers comprising SEQ ID NOS: 124 and 125 is provided.
  • This mixture is for the RT-PCR and primary PCR reactions for HIV. It also reverse transcribes and amplifies the HIV integrase region comprising positions 138, 140, 148, and 155.
  • a mixture of primers comprising SEQ ID NOS: 127 and 128. This mixture does not reverse transcribe or amplify the protease regions of interest, but is useful for the analysis of the reverse transcriptase.
  • oligonucleotide mixtures for use in the mutation- specific PCR reactions disclosed herein. Detection can be achieved so long as any of the disclosed forward primers are paired with any of the reverse primers for a given mutation.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS:22,23,24 and 25.
  • This is a forward primer mixture for the 103N mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer consisting of SEQ ID NO:26.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS: 59,60 and 61.
  • This is a forward primer mixture for the 103N mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer consisting of SEQ ID NO:26.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS:33,34 and 35 is provided.
  • This is a forward primer mixture for the 184V mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:36.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS:88, 89, 102, 103, and 104 is provided.
  • This is a forward primer mixture for the 184V mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:85.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS:62,63,64,65,96 and 97 is provided.
  • This is a forward primer mixture for the 41 L mutation- specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:66.
  • a mixture of primers comprising SEQ ID NOS: 10 and 98 and a reverse primer is provided.
  • This mixture includes a forward primer for the 65R mutation- specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO: 1 1.
  • a mixture of primers comprising SEQ ID NOS: 1 13 and a reverse primer is provided.
  • This mixture includes a forward primer for the 65R mutation- specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO: 1 14.
  • a mixture of primers comprising SEQ ID NOS: 1 17 and 1 18 and a reverse primer is provided.
  • This mixture includes a forward primer for the 65R mutation- specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO: 1 19.
  • a mixture of primers comprising SEQ ID NOS: 1 17, 1 18, 122, and a reverse primer is provided.
  • This mixture includes a forward primer for the 65R mutation- specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO:l 19.
  • a mixture of primers comprising SEQ ID NOS: 69 and 70 is provided. This is a forward primer mixture for the 67N mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:8.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS: 12, 13, and 71 is provided.
  • This is a forward primer mixture for the 69T specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:8 and 14.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS:2, 16,17,18,19, and 100 is provided.
  • This is a forward primer mixture for the 70R mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:20,72, or 73.
  • a mixture of primers comprising SEQ ID NOS:28 and 29 is provided. This is a forward primer mixture for the 181C mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:30.
  • a mixture of primers comprising SEQ ID NOS:83 and 84 is provided. This is a forward primer mixture for the protease 181C mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:85.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS:38,39,74,75, and 101 is provided.
  • This is a forward primer mixture for the 215T mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:45.
  • a mixture of primers comprising SEQ ID NO:40 and a reverse primer is provided.
  • This is a primer mixture for the 215Y mutation-specific PCR reaction.
  • the reverse primer can be, for example, a primer comprising or consisting of SEQ ID NO:45.
  • a mixture of primers comprising SEQ ID NO:41 and a reverse primer is provided.
  • This is a primer mixture for the 215F mutation-specific PCR reaction.
  • the reverse primer can be, for example, a primer comprising or consisting of SEQ ID NO:45.
  • a mixture of primers comprising SEQ ID NO:42 and a reverse primer is provided.
  • This is a primer mixture for the 215S mutation-specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO:45.
  • a mixture of primers comprising SEQ ID NO:43 and a reverse primer is provided.
  • This is a primer mixture for the 215C mutation- specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO:45.
  • a mixture of primers comprising SEQ ID NO:44 and a reverse primer is provided. This is a primer mixture for the 215D mutation-specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO:45.
  • a mixture of primers comprising SEQ ID NOS:48 and 49 is provided. This is a forward primer mixture for the protease 30N mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:50.
  • a mixture of primers comprising one or more primers selected from the group consisting of SEQ ID NOS:53,54,55,78,79 and 80 is provided.
  • This is a forward primer mixture for the protease 90M mutation-specific PCR reaction.
  • the mixture can further include a reverse primer.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:56 and 81.
  • a mixture of primers comprising SEQ ID NO: 133, and a reverse primer is provided.
  • This mixture includes a forward primer for the HIV-1 integrase 138K mutation-specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO: 130.
  • a mixture of primers comprising SEQ ID NO: 134, and a reverse primer is provided.
  • This mixture includes a forward primer for the HIV-1 integrase 140S mutation-specific PCR reaction.
  • the reverse primer can, for example, be a primer comprising or consisting of SEQ ID NO: 130.
  • a mixture of primers comprising SEQ ID NO: 137, and a forward primer is provided.
  • This mixture includes a reverse primer for the HIV-1 integrase 155H mutation-specific PCR reaction.
  • the forward primer can, for example, be a primer comprising or consisting of SEQ ID NO: 126.
  • a mixture of primers comprising SEQ ID NO: 138, and a forward primer is provided.
  • This mixture includes a reverse primer for the HIV-1 integrase 148R mutation-specific PCR reaction.
  • the forward primer can, for example, be a primer comprising or consisting of SEQ ID NO: 126.
  • a mixture of primers comprising SEQ ID NO: 139, and a forward primer is provided.
  • This mixture includes a reverse primer for the HIV-1 integrase 148H mutation-specific PCR reaction.
  • the forward primer can, for example, be a primer comprising or consisting of SEQ ID NO: 126.
  • mixtures of primers for mutation-specific PGR reaction for reverse transcriptase and protease can comprise a forward and reverse primer for a reverse transcriptase mutation and a forward and reverse primer for a protease mutation.
  • the forward primers in the mixture can include any forward primer for the specific RT mutation to be detected and any forward primer for the protease mutation to be detected.
  • These mixtures can be used to simultaneously detect both an RT mutation and a protease mutation.
  • An example of such a mixture of primers comprises or consists of SEQ ID NOS: 113, 1 17, 118, and 122.
  • the mixture can further include reverse primers.
  • the reverse primers can comprise or consist of SEQ ID NOS : 1 14, 1 19, and 81.
  • the mixtures (and methods) disclosed herein can utilize forward or reverse primers for other than those exemplified.
  • the exemp!ified mutation non-specific forward or reverse primers were found to work well.
  • the requirements of the mutation non-specific forward or reverse primer in the present method are typical of mutation non-specific forward or reverse primers designed and used routinely, and other mutation non-specific forward or reverse primers can be routinely made and used.
  • the mutation non-specific forward or reverse primer will be within about 40 to 250 bases from the mutation specific forward or reverse primer.
  • the mutation non-specific forward or reverse primer will be positioned in a stable location lacking a degree of variability that would impede binding.
  • the mutation non-specific forward or reverse primer is most likely to be located in the RT gene, the protease gene, or the integrase gene, but the exact location is routinely variable based on the usual criteria for mutation non-specific forward or reverse primer positioning.
  • Amplification mixtures are provided that include a probe for use in a real time PCR reaction.
  • the mixtures can thus include a forward primer, a reverse primer and a probe.
  • an amplification mixture is provided comprising a forward primer or a mixture of forward primers that amplifies the 103N, 65R, and 70R mutations and for 69T, wherein the mixture further comprises an
  • oligonucleotide having the nucleotides as set forth in SEQ ID NO:9, 1 15, 120, 121 , or combinations thereof. This is an example of a probe that can be used in any of these mutation-specific PCR reactions. This probe can also be used in the total copy PCR reaction.
  • An amplification mixture comprising a forward primer or a mixture of forward primers that amplifies the 41 L mutations, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NO:67.
  • This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification mixture comprising a forward primer or a mixture of forward primers that amplifies the 65R, and 67N mutations, and for 69T, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NO:68, 115, 116, 120, 121, or combinations thereof. This is an example of a probe that can be used in any of these mutation-specific PCR reactions.
  • An amplification mixture comprising a forward primer or a mixture of forward primers that amplifies the 70R mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:9 or 67.
  • This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the 181 C and 184V mutations, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NO:32. This is an example of a probe that can be used in either of these mutation- specific PCR reactions.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the 215 mutations, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:47, 76, or 77.
  • SEQ ID NOS:47, 76, or 77 are examples of probes that can be used in any of these mutation- specific PCR reactions.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the protease 3 ON mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NO:52.
  • This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the protease 90M mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:58 or 82.
  • This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the 103N mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NO: 9.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the 181 C mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:86 or 87.
  • SEQ ID NOS:86 or 87 are examples of probes that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the 184V mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:86, or 87.
  • SEQ ID NOS:86, or 87 are examples of probes that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the integrase 138K mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NO:
  • the probes of SEQ ID NO: 131 and 132 are optionally used in combination, optionally at a concentration percentage of 20% and 80% respectively.
  • probes that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the integrase 140S mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NO: 131 or 132.
  • the probes of SEQ ID NO: 131 and 132 are optionally used in combination, optionally at a concentration percentage of 20% and 80% respectively. These are examples of probes that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification mixture comprising a forward primer or mixture of forward primers that amplifies the integrase 155H mutation, wherein the mixture further comprises an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:140 or 141.
  • the probes of SEQ ID NO: 140 and 141 are optionally used in combination, optionally at a concentration percentage of 80% and 20% respectively.
  • probes that can be used in mutation-specific PCR reactions for this mutation.
  • the probe can incorporate a detectable moiety.
  • detectable moiety is intended to mean any suitable label, including, but not limited to, enzymes, fluorophores, biotin, chromophores, radioisotopes, colored particles, electrochemical, chemical-modifying or chemiluminescent moieties. Examples include (i) enzymes which can catalyze color or light emitting (luminescence) reactions and (ii) fluorophores.
  • the detection of the detectable moiety can be direct provided that the detectable moiety is itself detectable, such as, for example, in the case of fluorophores. Alternatively, the detection of the detectable moiety can be indirect.
  • a second moiety readable with the detectable moiety, itself being directly detectable is preferably employed.
  • the detectable moiety may be inherent to a molecular probe.
  • Common fluorescent moieties include: fluorescein, cyanine dyes, coumarins, phycoerythrin, phycobiliproteins, dansyl chloride, Texas Red, and lanthanide complexes.
  • the size of the primers or probes for interaction with the nucleic acids can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification or the simple hybridization of the probe or primer.
  • a typical primer or probe would be at least, less than or equal to 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
  • the primers for the reverse transcriptase gene, protease gene, or integrase gene typically will be used to produce an amplified DNA product that contains a region of the reverse transcriptase gene, protease gene, or integrase gene containing the relevant site(s) of the mutation(s) of interest.
  • typically the size of the product will be such that the size can be accurately determined to within 3, or 2 or 1 nucleotides.
  • This product can be at least, less than or equal to 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800
  • a polynucleotide comprising naturally occurring nucleotides and
  • phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template.
  • a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et ah, supra, 1995,
  • the nucleic acids such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition
  • Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al., Bioconjug. Chem. 5:3-7 (1994), incorporated herein by reference.
  • kits that are drawn to reagents that can be used in practicing the methods disclosed herein.
  • the kits can include any reagent or combination of reagents discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
  • the kits could include primers to perform the amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended. Specific guidance as to the components of the kits is provided herein, including buffers, primers and probes.
  • kits for detecting a mutation in the reverse transcriptase gene or protease gene of HIV comprising one or more of the oligonucleotides set forth in SEQ ID Nos:l-92, 1 14- 122, or 124-141, or any portion thereof.
  • HIV-1 protease and an HIV-1 reverse transcriptase are provided below. Also provided are accession numbers for these and other HIV-1 protease and an HIV-1 reverse transcriptase coding sequences. Accession numbers for amino acid sequences of the HIV-1 reverse transcriptase and the HIV-1 protease are also provided. This information, along with sequence information on many more examples of HIV-1 protease and reverse transcriptase proteins and coding sequences, are in the art. As such, they constitute a part of the disclosure of the present application.
  • RNA can be used in the present invention.
  • R A is not limited to that obtained from plasma or serum, but can also be intracellular RNA that has not been packaged. Detection can be achieved so long as any of the disclosed primers are paired with any of the mutation specific forward or reverse primers for a given mutation. The following methods describe specific sets of primers that achieve especially sensitive levels of detection.
  • HIV-1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:6 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer set selected from the group consisting of SEQ ID NOS: 1 ,2,4 and 5 to produce a DNA product; and (c) contacting the DNA product of step (b) with a reverse primer and a primer set selected from the group consisting of SEQ ID NOS:22,23,24 and 25 and SEQ ID NOS:59,60 and 61 to amplify HIV-1 DNA containing the 103N mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:26.
  • the presence of an amplification signal within a certain number of cycles after signal detection in the total copy PCR reaction indicates the presence of the respective mutation.
  • This method for use with an RNA template, detects the 103N mutation in either or both of Subtype B and Subtype C.
  • SEQ ID NOS: 4 and 5 are forward RT-PCR (for RNA) and primary PCR (for DNA) primers for Subtype C.
  • SEQ ID NO:4 includes protease sequences while SEQ ID NO:5 is for reverse transcriptase only.
  • SEQ ID NOS: 1 and 2 are forward RT-PCR (for RNA) and primary PCR (for DNA) primers for Subtype B.
  • SEQ ID NO:l includes protease sequences while SEQ ID NO:2 is for reverse transcriptase only.
  • step (c) of these methods a set of primers is used, including at least a primer pair comprising a reverse primer and one of the disclosed forward primers for the respective mutation.
  • step (b) of the methods starting with RNA the choice of amplifying both the reverse transcriptase and the protease are provided by an exemplary primary PCR forward primer that includes protease and an exemplary primary forward primer for reverse transcriptase only.
  • Each forward primer disclosed for the RT-PCR reaction or the primary PCR reaction in the methods disclosed works independently. If a protease analysis is to be done, then the Fl primers must be used for the RT-PCR or primary PCR steps.
  • Reverse transcriptase analyses can be performed from the F2+reverse primer products alone (the F2 primers are slightly more sensitive than the Fl primers, thus can provide the user with a more sensitive test).
  • step (b) of the methods starting with RNA there is reverse primer remaining in the reaction product from step (a).
  • the RT step of the present methods can utilize RT primers other than those described. The only requirement is that the primers generate a template in the relevant region of the reverse transcriptase gene or in the protease gene or both.
  • a further method for detecting the 103N mutation in the reverse transcriptase of HIV- 1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l ,2,4 and 5 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a reverse primer and a primer set selected from the group consisting of SEQ ID NOS:22,23,24 and 25 and SEQ ID NOS;59,60 and 61 to amplify HIV-1 DNA containing the 103N mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:26. This method, for use with a DNA template, detects the 103N mutation in either or both of Subtype B and Subtype C.
  • step (b) of these methods a set of primers is used, including at least a primer pair comprising a reverse primer and one of the disclosed forward primers for the respective mutation.
  • step (a) of the methods starting with DNA the choice of amplifying the reverse transcriptase, the protease, and the integrase are provided by an exemplary primary PCR forward primer that includes protease and an exemplary forward primer for reverse transcriptase, and a forward primer for integrase only.
  • Each forward primer disclosed for the primary PCR reaction in the method beginning with DNA works independently.
  • the RT-only primer and the protease-included primer can be used independently with a reverse primer. If a protease analysis is to be done, then the Fl primers must be used for the RT-PCR or primary PCR steps. Reverse transcriptase analyses can be performed from the F2+reverse primer products alone (the F2 primers are slightly more sensitive than the Fl primers, thus can provide the user with a more sensitive test).
  • Amplification methods are provided that include a probe for use in a real time PCR reaction.
  • the methods can thus include the use of a forward primer, a reverse primer and a probe.
  • an amplification method is provided comprising a forward primer or a mixture of forward primers that amplifies the protease 90M, and the reverse transcriptase 103N, 65R, and 70R mutations, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NO:9.
  • This is an example of a probe that can be used in any of these mutation- specific PCR reactions. This probe can also be used in the total copy PCR reaction.
  • transcriptase of HIV- 1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:33,34 and 35 and a reverse primer to amplify
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:36,
  • transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS:33,34 and 35 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 184V mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:36.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype B 181C and Subtype B 184V mutations, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NO:32. This is an example of a probe that can be used in either of these mutation-specific PCR reactions.
  • a method for detecting a Subtype B 41 L mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:62,63,64 and 65 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 41L mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:66.
  • a method for detecting a Subtype B 41 L mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing R A extracted from HIV- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:63 ,96,97,64, and 65 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 41 L mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:66.
  • a method for detecting a Subtype B 41L mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 62,63,64 and 65 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 41 L mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:66.
  • a method for detecting a Subtype B 41 L mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 63,96,97,64, and 65 and a reverse primer to amplify HiV-l DNA containing a Subtype B 41 L mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:66.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype B 41 L mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NO:67. This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • a method for detecting a Subtype B 65R mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV- 1 with a primer comprising SEQ ID NO: 3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer comprising SEQ ID NO: 10 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 65R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:l 1.
  • a method for detecting a Subtype B 65R mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer comprising SEQ ID NO:98 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 65R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 11.
  • a method for detecting a Subtype B 65R mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer comprising SEQ ID NO: 10 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 65R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 1 1.
  • a method for detecting a Subtype B 65R mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer comprising SEQ ID NO:98 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 65R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 1 1.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype B 65R mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:9, 68, or 99. These are examples of probes that can be used in mutation- specific PCR reactions for this mutation.
  • a method for detecting a Subtype AE 65R mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a common DNA amplification product; and (b) contacting the DNA of step (a) with a primer comprising SEQ ID NO: l 13 and a reverse primer to amplify HIV-1 DNA containing a Subtype AE 65R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 114.
  • a method for detecting a Subtype C 65 mutation in the reverse transcriptase of HIV- 1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: 1 and 2 produce a common DNA amplification product; and (b) contacting the DNA of step (a) with a primer comprising SEQ ID NOs:l 17 and 1 18, and a reverse primer to amplify HlV-1 DNA containing a Subtype C 65R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 1 19.
  • a method for detecting a Subtype C 65R mutation in the reverse transcriptase of HIV- 1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a common DNA amplification product; and (b) contacting the DNA of step (a) with a primer comprising SEQ ID NOs: l 17, 118, and 122, and a reverse primer to amplify HIV-1 DNA containing a Subtype C 65R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 119.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype AE 65R mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NOS: 115, 116, or combinations thereof. These are examples of probes that can be used in mutation-specific PCR reactions for this mutation.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype C 65R mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NOS: 120, 121, or combinations thereof. These are examples of probes that can be used in mutation-specific PCR reactions for this mutation.
  • a method for detecting a Subtype B 67N mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:69 and 70 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 67N mutation.
  • the reverse primer is routinely selected based on the well- known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:8.
  • a method for detecting a Subtype B 67N mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS:69 and 70 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 67N mutation.
  • the reverse primer is routinely selected based on the well- known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:8.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype B 67N mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NO:68. This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • a method for detecting a Subtype B 69T in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: l and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS: 12 and 13 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 69T.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 14.
  • a method for detecting a Subtype B 69T in the reverse transcriptase of HIV- 1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS: 12 and 71 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 69T.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:8.
  • a method for detecting a Subtype B 69T in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 12 and 13 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 69T.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 14,
  • a method for detecting a Subtype B 69T in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 12 and 71 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 69T.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:8.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype B 69T, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:9 or 68. These are examples probes that can be used in mutation-specific PCR reactions for this mutation.
  • a method for detecting a Subtype B 70R mutation in the reverse transcriptase of HIV- 1 comprising (a) reverse transcribing RNA extracted from H1V- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS: 16,17,18 and 19 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 70R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:20.
  • a method for detecting a Subtype B 70R mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NO:2 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 70R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:72 and 73.
  • a method for detecting a Subtype B 70R mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NO: 100 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 70R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:72 and 73.
  • a method for detecting a Subtype B 70R mutation in the reverse transcriptase of HIV- 1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 16, 17, 18 and 19 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 70R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:20.
  • a method for detecting a Subtype B 70R mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NO:2 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 70R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:72 and 73.
  • a method for detecting a Subtype B 70R mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l and 2 to amplify the
  • step (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NO: 100 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 70R mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:72 and 73.
  • An amplification method is provided comprising a forward primer or mixture of forward primers that amplifies a Subtype B 70R mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NO:9 or 67. This is an example of a probe that can be used in mutation- specific PCR reactions for this mutation.
  • transcriptase of HIV- 1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and (c) contacting the DN A product of step (b) with a primer set comprising SEQ ID NOS:22,23,24 and 25 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 103N mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:26.
  • transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS:22,23,24 and 25 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 103N mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:26.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype B 103N mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NO:9. This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • transcriptase of HIV- 1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:28 and 29 and a reverse primer to amplify HIV- 1 DNA containing a Subtype B 181C mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:30.
  • transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 28 and 29 and a reverse primer to amplify HIV-1 DNA containing a Subtype B 181C mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:30.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype B 181C mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NO:32. This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • a method for detecting a Subtype B 215T mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO: 3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 or SEQ ID NOS:74 and 75 or SEQ ID NOS:101 and 75 to produce a DNA product; (c) contacting the DNA product of step (b) with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:38 and 39 to amplify HIV-1 DNA containing a Subtype B 215 mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO :45.
  • a method for detecting a Subtype B 215 mutation in the reverse transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; (c) contacting the DNA product of step (b) with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:40,41,42,43 and 44 to amplify HIV-1 DNA containing a Subtype B 215 mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:45.
  • a method for detecting a Subtype B 215 mutation in the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:40,41 , 42,43 and 44 to amplify HIV-1 DNA containing a Subtype B 215 mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:45.
  • any or all of the Y, F, S, C or D mutations can be detected.
  • the forward primers can be used together in the reaction mixture.
  • the forward primer for that mutation would be used alone.
  • Specific combinations of mutations at 215 can be identified by using the desired subset of the disclosed forward primers.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies Subtype B 215 mutations, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:47, 76, or 77. This is an example of a probe that can be used in mutation- specific PCR reactions for this mutation.
  • a method for detecting the 30N mutation in the protease of HIV- 1 Subtype B comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product;
  • step (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; and
  • step (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:48 and 49 and a reverse primer to amplify HIV-1 DNA containing the 30N mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 50.
  • a method for detecting the 30N mutation in the protease of HIV-1 Subtype B comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: land 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS:48 and 49 and a reverse primer to amplify HIV-1 DNA containing the 30N mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:50.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies the protease 30N mutation of HIV-1 Subtype B, wherein the method further comprises the use of an oligonucleotide having the nucleotides as set forth in SEQ ID NO:52. This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • a method for detecting the 90M mutation in the protease of HIV- 1 Subtype B comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO: 3 to produce a reverse transcription reaction product;
  • step (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:l and 2 to produce a DNA product; and
  • step (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:53,54, and 55 and a reverse primer to amplify HIV-1 DNA containing the 90M mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:56.
  • a method for detecting the 90M mutation in the protease of HIV-1 Subtype B comprising (a) reverse transcribing RNA extracted from HIV- 1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product;
  • step (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and
  • step (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS: 55,78,79, and 80 and a reverse primer to amplify HIV-1 DNA containing the 90M mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 81.
  • a method for detecting the 90M mutation in the protease of HIV-1 Subtype B comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS:53,54, and 55 and a reverse primer to amplify HIV-1 DNA containing the 90M mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:56.
  • a method for detecting the 90M mutation in the protease of HIV-1 Subtype B comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS:55,78,79, and 80 and a reverse primer to amplify HIV-1 DNA containing the 90M mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 81.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies the protease 90M mutation, wherein the method further comprises the use of an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:58 or 82. These are examples probes that can be used in mutation- specific PC reactions for this mutation.
  • transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:6 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:3 and 4 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:59,60, and 61 and a reverse primer to amplify HIV-1 DNA containing a Subtype C 103N mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:26.
  • transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 59,60, and 61 and a reverse primer to amplify HIV-1 DNA containing a Subtype C 103N mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:26.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype C 103N mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NO:9. This is an example of a probe that can be used in mutation-specific PCR reactions for this mutation.
  • transcriptase of HIV- 1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:6 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:3 and 4 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:83 and 84 and a reverse primer to amplify HIV- 1 DNA containing a Subtype C 181 C mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:85.
  • transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:3 and 4 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS:83 and 84 and a reverse primer to amplify HIV-1 DNA containing a Subtype C 181C mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:85.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype C 103N mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:86 or 87. These are examples of probes that can be used in mutation-specific PCR reactions for this mutation.
  • transcriptase of HIV- 1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:6 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:3 and 4 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:88 and 89 and a reverse primer to amplify HIV- 1 DNA containing a Subtype C 184V mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:85.
  • transcriptase of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO: 6 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS:3 and 4 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS: 102,103 and 104 and a reverse primer to amplify HIV-1 DNA containing a Subtype C 184V mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:85.
  • transcriptase of HIV- 1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS:l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 88 and 89 and a reverse primer to amplify HIV-1 DNA containing a Subtype C 184V mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:85.
  • transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: l and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS: 102,103 and 104 and a reverse primer to amplify HIV-1 DNA containing a Subtype C 184V mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 85.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies a Subtype C 184V mutation, wherein the method further comprises using an oligonucleotide having the nucleotides as set forth in SEQ ID NOS:86 or 87. These are examples of probes that can be used in mutation-specific PCR reactions for this mutation.
  • a method for detecting al 38K mutation in the integrase of HIV - 1 comprising (a) contacting DNA with a reverse primer and a forward primer selected from the group consisting of SEQ ID NOS: 126 and 129 to produce a common DNA amplification product; and (b) contacting the DNA of step (a) with a mutation specific forward primer comprising SEQ ID NOS: 133 and a reverse primer to amplify HIV-1 DNA containing a integrase 138K mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 130.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies an integrase 138K mutation, wherein the method further comprises oligonucleotides having the nucleotides as set forth in SEQ ID NOS: 131 , 132 or combinations thereof.
  • the oligonucleotides of SEQ ID NO: 131 and 132 are optionally used in combination optionally at a concentration percentage of 20% and 80% respectively. These are examples of probes that can be used in mutation-specific PCR reactions for this mutation.
  • a method for detecting al40S mutation in the integrase of HIV-1 comprising (a) contacting DNA with a reverse primer and forward primer selected from the group consisting of SEQ ID NOS: 126 and 129 to produce a common DNA amplification product; and (b) contacting the DNA of step (a) with a mutation specific forward primer comprising SEQ ID NO: 134 and a reverse primer to amplify HIV-1 DNA containing a integrase 140S mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 130.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies an integrase 140S mutation, wherein the method further comprises using oligonucleotides having the nucleotides as set forth in SEQ ID NOS: 131 , 132 or combinations thereof.
  • the oligonucleotides of SEQ ID NO: 131 and 132 are optionally used in combination at a concentration percentage of 20% and 80% respectively. These are examples of probes that can be used in mutation- specific PCR reactions for this mutation.
  • a method for detecting al55H mutation in the integrase of HIV-1 comprising (a) contacting DNA with a forward primer and a reverse primer selected from the group consisting of SEQ ID NOS: 124 and 129 to produce a common DNA amplification product; and (b) contacting the DNA of step (a) with a mutation specific reverse primer comprising SEQ ID NO: 137 and a forward primer to amplify HIV-1 DNA containing a integrase 155H mutation.
  • the forward primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 126.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies an integrase 155H mutation, wherein the method further comprises using oligonucleotides having the nucleotides as set forth in SEQ ID NOS: 140, 141 or combinations thereof.
  • the oligonucleotides of SEQ ID NO: 140 and 141 are optionally used in combination at a concentration percentage of 80% and 20% respectively. These are examples of probes that can be used in mutation- specific PCR reactions for this mutation.
  • a method for detecting al48R mutation in the integrase of HIV- 1 comprising (a) contacting DNA with a forward primer and a reverse primer selected from the group consisting of SEQ ID NOS: 124 and 125 to produce a common DNA amplification product; and (b) contacting the DNA of step (a) with a mutation specific reverse primer comprising SEQ ID NOS: 138 and a forward primer to amplify HIV-1 DNA containing a integrase HSR mutation.
  • the forward primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 126.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies an integrase 148R mutation, wherein the method further comprises using oligonucleotides having the nucleotides as set forth in SEQ ID NOS: 140, 141 or combinations thereof.
  • the oligonucleotides of SEQ ID NO: 140 and 141 are optionally used in combination at a concentration percentage of 80% and 20% respectively. These are examples of probes that can be used in mutation- specific PCR reactions for this mutation.
  • a method for detecting al48H mutation in the integrase of HIV-1 comprising (a) contacting DNA with a forward primer and a reverse primer selected from the group consisting of SEQ ID NOS: 124 and 125 to produce a common DNA amplification product; and (b) contacting the DNA of step (a) with a mutation specific reverse primer comprising SEQ ID NOS: 139 and a forward primer to amplify HIV-1 DNA containing a integrase 148H mutation.
  • the forward primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 126.
  • An amplification method comprising a forward primer or mixture of forward primers that amplifies an integrase 148H mutation, wherein the method further comprises using oligonucleotides having the nucleotides as set forth in SEQ ID NOS: 140, 141 or combinations thereof.
  • the oligonucleotides of SEQ ID NO: 140 and 141 are optionally used in combination at a concentration percentage of 80% and 20% respectively. These are examples of probes that can be used in mutation- specific PCR reactions for this mutation.
  • a method for amplifying the reverse transcriptase of HIV- 1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO: 3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer comprising SEQ ID NO:7 and a reverse primer to amplify a region encoding the reverse transcriptase of HIV-1.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO:8.
  • This can be a common amplification method of the invention.
  • the total copy reaction can be used to provide the baseline for the mutation-specific real time PCR reactions disclosed herein.
  • matched wildtype primers can be used as a control, as is known to one skilled in the art.
  • a method for amplifying the reverse transcriptase of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: 1 and 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer comprising SEQ ID NO:7 and a reverse primer to amplify a region encoding the reverse transcriptase of HIV-1.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NO: 8.
  • the amplification methods disclosed herein can utilize reverse primers other than those exemplified.
  • the exemplified reverse primers were found to work well. However, the requirements of the reverse primer in the present method are typical of reverse primers designed and used routinely, and other reverse primers can be routinely made and used. It is expected that the reverse primer will be within about 40 to 250 bases from the forward primer. It is also expected that the reverse primer will be positioned in a stable location lacking variability to a degree that would impede binding. The reverse primer is most likely to be located in the RT gene or the protease gene, but the exact location is routinely variable based on the usual criteria for reverse primer positioning.
  • Methods disclosed herein can further include detection, in the same mixture, of a specified RT mutation, a specified protease mutation, and/or a specified integrase mutation.
  • the methods described herein are interchangeable and combinable depending on the desire of the user to detect one or a plurality of mutations in one or more HIV-1 sequences.
  • a method for detecting a 184 V mutation in the reverse transcriptase of HIV-1 and a 90M mutation in the protease of HIV-1 comprising (a) reverse transcribing RNA extracted from HIV-1 with a primer comprising SEQ ID NO:3 to produce a reverse transcription reaction product; (b) contacting the reverse transcription product of step (a) with a primer selected from the group consisting of SEQ ID NOS: l and 2 to produce a DNA product; and (c) contacting the DNA product of step (b) with a primer set comprising SEQ ID NOS:33,34, 35, 55, 78,79, and 80 and a reverse primer to amplify HIV-1 DNA containing a 184V and a 90M mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:36 and 81.
  • a method for detecting a 184V mutation in the reverse transcriptase of HIV-1 and a 90M mutation in the protease of HIV-1 comprising (a) contacting DNA with a reverse primer and a primer selected from the group consisting of SEQ ID NOS: land 2 to amplify the DNA; and (b) contacting the amplified DNA of step (a) with a primer set comprising SEQ ID NOS:33,34,35,55,78,79, and 80 and a reverse primer to amplify HIV-1 DNA containing a 184V and a 90M mutation.
  • the reverse primer is routinely selected based on the well-known criteria for such selections, which are described herein and elsewhere.
  • the reverse primer can be a primer comprising or consisting of SEQ ID NOS:36 and 81.
  • a variety of dyes are known to exhibit increased fluorescence in response to binding double stranded DNA. Production of wild type or mutation containing PCR products are continuously monitored by the increased fluorescence of dyes such as ethidium bromide or Syber Green as they bind to the accumulating PCR product. Note that dye binding is not selective for the sequence of the PCR product, and high non-specific background can give rise to false signals with this technique.
  • exonuclease primers e.g., TaqMan® probes
  • exonuclease primers utilizes the 5 1 exonuclease activity of thermostable polymerases such as Taq to cleave dual-labeled probes present in the amplification reaction (Wittwer, C. et al. Biotechniques 22:130-138, 1 97; Holland, P et al PNAS 88:7276-7280, 1991, incorporated herein by reference).
  • the probes used in this assay are distinct from the PCR primer and are dually-labeled with both a molecule capable of fluorescence and a molecule capable of quenching fluorescence.
  • An additional form of real-time PCR also capitalizes on the intramolecular quenching of a fluorescent molecule by use of a tethered quenching moiety.
  • the molecular beacon technology utilizes hairpin-shaped molecules with an internally- quenched fluorophore whose fluorescence is restored by binding to a DNA target of interest (Kramer, R. et al. Nat. Biotechnol. 14:303-308, 1996, incorporated herein by reference). Increased binding of the molecular beacon probe to the accumulating PCR product can be used to specifically detect SNPs present in genomic DNA.
  • a final, general fluorescent detection strategy used for detection of point mutations and SNP in real time utilizes synthetic DNA segments known as hybridization probes in conjunction with a process known as fluorescence resonance energy transfer (FRET) (Wittwer, C. et al. Biotechniques 22: 130-138, 1997; Bernard, P. et al. Am. J. Pathol. 153:1055-1061, 1998, incorporated herein by reference).
  • FRET fluorescence resonance energy transfer
  • selective hybridization conditions can be defined as stringent hybridization conditions.
  • stringency of hybridization is controlled by both
  • the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies.
  • Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations.
  • the conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987: 154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids, which are incorporated herein in their entireties).
  • a preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C.
  • Stringency of hybridization and washing can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
  • This example describes the development and application of real-time PCR- based point mutation assays for the 103N and 184V mutations in the reverse transcriptase (RT) of HIV-1.
  • the assay measures the differential amplifications of total copy and mutation- specific reactions that target codons of interest.
  • mutation-containing plasmids diluted in backgrounds of wild type plasmid
  • the assays were able to achieve a mutation detection limit of 0.04% and 0.2% 103N and 184V, respectively.
  • To evaluate the performance of these assays with clinical specimens 77 known wild-type samples were first analyzed. None of the wild-type samples was positive for the 184V mutation, while one sample (1.3%) scored positive for 103N.
  • 103N was detected in 54 of 55 positive specimens (98%), and 184V in 65 of 67 (97%).
  • the present assays were applied to a test population of HIV-1 -positive treatment-na ' ive persons documented to have RT mutations other than 103N and 184V.
  • the 103N assay detected 4 positive samples (2.4%) in 165 plasma samples previously found absent of 103N (clones are currently being screened for the presence of low-level mutants).
  • 173 samples previously determined to be negative for the 184V mutation three samples scored positive (1.1%) for 184V by this assay.
  • Wild type HIV-1 subtype B samples were obtained from the plasma of 23 treatment-na ' ive persons (2) with no detectable resistance mutations, and from 54 sera collected in the early 1980's, prior to the development of antiretroviral drugs. 67 specimens confirmed by sequence analysis to have virus carrying the mutation comprised mutation-positive samples. The test population encompassed a second group of 173 treatment-naive patients (partially referenced in 2), all with RT mutations other than 103N or 184 V. Approximately 17% of the treatment-naive specimens were from persons documented to be recently infected. Results obtained from evaluation of the wild type and mutation-confirmed samples were used to define the assay cutoffs.
  • HIV-1 genomic RNA was extracted (Qiagen UltraSens RNA kit) from patient plasma or serum. Primary amplifications of HIV-1 template were generated by reverse transcriptase-PCR using primers that demarcated the first half of the RT sequence, or when desired, the forward primer was shifted upstream to also include the entire protease region. The minimum copy numbers from which these reactions could successfully amplify were 5 and 10 RNA copies, respectively.
  • Baseline measurements for viral copies in test samples were determined using HIV-1 RT total copy primers with a total copy probe (Fig. 1 A).
  • Preliminary analysis for detecting the presence of the mutation was performed using primer mixtures to compensate for polymorphic variability in the primer binding sites.
  • the 103N- specific mixture incorporated four different primers, and the 184V-specific reaction used three primers.
  • the primers can be mixed at optimal ratios to equilibrate the differences in primer affinities. Examples of such ratios are provided below. It is recognized that the optimization of primer ratios is routine given the teaching of the primers and primer mixtures themselves. One of skill can envision alternative ratios.
  • the mutation-specific primers were designed to maximize specificity for annealing to the mutated nucleotide(s), thus having a reduced affinity for wild type sequences (3,4).
  • the probes for each reaction were 5'labeled with FAM and quenched with QSY-7.
  • the choice of fluorophore and quencher can be routinely varied. Common fluorophores include HEX, ROX, Texas Red, TAMRA, JOE, Cy3, Cy5, S YBR and VIC. There are others that often overlap the above spectra and can be used.
  • the Bio-Rad fluorophore table contains a more complete listing of fluorophores that can be used for this method.
  • Degradation of the fluorescent probes during chain elongation removes the fluorophore from the proximity of the quencher and generates the fluorescent signals, reported as relative fluorescent units (RFU), that increase with each amplification cycle (Fig. IB).
  • the cycle number where the fluorescence emission exceeds the software-derived threshold is called the threshold cycle (CT) and is the unit of measure when comparing the differences in amplification levels (ACT) of the total copy and mutation-specific reactions.
  • Assay detection limits were tested against dilutions of mutation-containing plasmid clones and from PCR products from both lab-adapted HlV- 1 and patient- derived mutant virus spiked into a background of wild type virus template.
  • the amounts of mutant input comprised 100%-0.001 % of the total virus population.
  • the 100 ⁇ primary PCR reaction may be diluted (1 : 10-1 :20) prior to the real-time PCR reaction to ensure the secondary reaction is not overloaded with template and to provide sufficient template for future studies.
  • a sequence-specific probe labeled with a fluorophore and a quencher, anneals to a region flanked by locus-specific primers. PCR extension from the primers degrades the intervening probe releasing the quencher from the proximity of the fluorophore, thus increasing the level of detectable fluorescence.
  • the amplification cycle at which the level of product (i.e., amount of degraded probe) is measurable above background, is the threshold cycle (TC). This value directly correlates with the amount of starting template and is the unit of measure when making comparisons between amplification levels.
  • the mutation-specific tests(c) can be qualitative, by comparing to the
  • the plasmid standards can be prepared and aliquotted as 40-cycle reactants of which 2 are used for the copy standards in each secondary reaction plate.
  • An advantage of the present invention is in detection sensitivity of the various subtypes of HIV from various countries of the world.
  • the sets disclosed in the primer set below are particularly sensitive to detection of HIV subtypes across the spectrum of HIV.
  • ReTi-HIV Assay Oligonucleotide list a Primers for the RT-PCR reaction:
  • RTPF 1 (includes protease) 5'-CCT CAG ATC ACT CTT TGG CAA CG
  • RTPF 2 (only RT) 5 '-AAA GTT AAA CAA TGG CCA TTG ACA G
  • RTPF 1C (includes protease) 5'-CCT CAA ATC ACT CTT TGG CAG CG
  • RTPF2C (RT only) 5'-AGG TTA AAC AAT GGC CAT TGA CAG AAG
  • Primers below are for the listed mutations. All forward primers for each mutation can be mixed for general surveillance testing or the primers can be used individually or mixed and matched for detecting/monitoring distinct polymorphisms associated with that mutation.
  • the primer proportions exemplified for these mixtures are routinely adjustable using the optimization methods routinely practiced in this field.
  • FAM 6-carboxy fluorescein, however, any fluorophore may be used;
  • HIV-AE K65R I F 5 ' - ATA YA AT ACTCC ARTATTTGCT ATAA AC A G (SEQ ID NO: 113) HIV-B ComRev 5'-TGG TGT CTC ATT GTT TRT ACT AGG TA (SEQ ID NO: 1 14
  • HIV-C 65 IP 5 * - V4JW-TCAGGGARC"T” , CAATAAAAGAACTCAAGACTTYTGGGA (SEQ ID NO: 1
  • HIV-C 6.
  • TAG RAC (SEQ ID NO: 86)
  • TAG RAC (SEQ ID NO: 87)
  • TAG RAC (SEQ ID NO: 86)
  • TAG RAC (SEQ ID NO: 86)
  • TAG RAC (SEQ ID NO: 87)
  • HIV-IN GEN.3F GTG ATA AAT GTC ARY TAA AAG GRG AAG C (SEQ ID NO: 124)
  • RT- PCR For RT- PCR
  • HIV-IN GEN .3R CTT TCC AAA STG GRT CTC TGC TG : (SEQ ID NO: 325)
  • CAT GGA CAA GTA GAC TGT AGY CCA SEQ ID NO: 126) : forward primer for common reaction and 148 and 1 55 mutations.
  • HIV-IN COM.1BR CAG CYT GMT CTC TTA CYT GTC CTA (SEQ ID NO: 129): used in common reaction with GEN .4F
  • HIV-B-1N 138.2P TCA AGC TGA ACA TC"T" YAA RAC AGC AGT ACA RAT GGC (SEQ ID NO: 131 ):
  • H1V-B-1N 138.
  • IP TCA GGC TGA ACA TCT" YAA RAC AGC AGT ACA RAT GGC (SEQ ID NO: 132): 138 probes used for 138 and 140 mutations
  • HIV-B IN 138K.1 F GTG GGC RGG RAT CAA RCT GA (SEQ ID NO: 133)
  • HIV-B IN 140S.1F, GGC RGG RAT CAA GCA GRA ATC TA (SEQ ID NO: 134) HIV-B-IN SEQ.3F, GTA GCC AGT GGA TAY ATA GAA GCA G (SEQ ID NO: 135):
  • HIV-B-IN 155H.1 R ACC TGT CCT ATA ATT TTC TTT AAT TCY TTC TG (SEQ ID NO: 137) HIV-B-IN 148R.1 R, CTT TAT TCA TAG ATT CTA CTA CYC GTC (SEQ ID NO: 138) HIV-B-IN 148H.2R, CTT TAA TTC TTT ATT CAT AGA TTC TAC TAC YCG A (SEQ ID NO: 139)
  • HIV-B IN COM.4.3P TCT TAA AAT "T”AG CAG GAA GAT GGC CAG TRA MAA CAA TAC ATA C (SEQ ID NO: 140)
  • HIV-B IN COM.4.4P TTT TAA AAC "T”AG CAG GAA GAT GGC CAG TRA MAA CAA TAC ATA C (SEQ ID NO: 141 )
  • COM probes are used for common reaction and 148 and 155 mutation tests.
  • nucleotides marked within quotation marks are optionally bound to a quencher molecule.
  • Probes are optionally labeled at the 5 ' nucleotide with a fluorophore that is matched to a quencher molecule.
  • Simian Immunodeficiency virus has strong clinical, pathological, virological and immunological analogies with HIV infection of humans. Infection of macaques with SIV provides a valuable model for exploring crucial issues related to both the pathogenesis and prevention of HIV infection. The model offers a unique setting for mutation detection testing, preclinical evaluation of drugs, vaccines and gene-therapies against HIV, and can identify many virus and host determinants of lentiviral disease.
  • the present invention can be utilized in conjunction with SIV nucleotide sequences.
  • SIV nucleotide sequences Provided below are exemplary SIV sequences for use with the present invention.
  • the SIVmac 65R mutation-specific reaction can be compared against the total copy (common) reaction in the same way as described previously for HIV oligonucleotides.
  • the following is a list of mutation specific primers with an example of the ratios/proportions of these primers that can be used to specifically and sensitively detect the respective mutations.
  • HIV-C 65.2P (20%) (SEQ ID NO: 121)
  • HIV-B-IN 138.2P (20%) (SEQ ID NO: 131)
  • IP (80%) (SEQ ID NO: 132)
  • HIV-B-IN 138.2P (20%) (SEQ ID NO: 131 )
  • HIV-B IN COM.4.4P (20%) (SEQ ID NO: 141)
  • HIV-B IN COM.4.4P (20%) (SEQ ID NO: 141)
  • HIV-B IN COM.4.4P (20%) (SEQ ID NO: 141)
  • the mixed-primer assay for 103N was able to distinguish as little as 0.04% 103N in within a wild type background.
  • the assay for 184V yielded discernable CTs for 184V plasmids when comprising as little as 0.2% of the population (Fig. 2A).
  • the 184V assay did not detect this mutation in any of the 77 documented wild type samples. However, with the 103N assay, 1 wild type sample scored positive (ACT of 10.6) for the mutation. The 103N discordant result might be signifying a very low level ( ⁇ 5%) naturally occurring polymorphism. The assay for the 103N mutation was able to detect the mutation in all 23 samples documented to have the mutation. The 184V assay was unable to detect the mutation in one (ACT of 9.8) of the 36 specimens known to have the mutation, yielding an assay sensitivity of 97.2%. This outlier was obtained from a treatment-experienced person having a mixed virus population with five polymorphisms in the primer binding site.
  • assay cutoffs of 0.2-0.5% mutant virus were used for screening purposes.
  • the sensitivities and specificities of the assays on genotyped clinical samples carrying the mutations of interest were found to range between 95-99%.
  • Real-time PCR screening of the 147 transmitted HIV-1 carrying resistance-related mutations detected additional mutations that expanded the spectrum of drugs to which the viruses were resistant.
  • the added mutants increased the prevalence of 90M from 8% to 10% (+25%), of 184V from
  • the present real-time PCR primer-mix point mutation assay for the HIV-1 103N and 184V RT mutation were able to detect as little as 0.04% and 0.2% mutant virus, respectively, in HIV-1 plasmid dilutions.
  • the primer designs were robust and worked well with the very high sequence variability in the clinical specimens examined.
  • the ACTs of the mutation-positive specimens formed a distinct cluster from the wild type samples and samples with other mutations. These assays have shown acceptable performance on 282 samples of plasma-derived HIV-1, providing a sensitivity of 97.2-100% and a specificity of 98.6%.
  • the benefits of real-time PCR-based testing include the following: 1) The real-time reaction requires a one-step setup, decreasing the potential for user error; 2) High throughput: reactions performed in 96-well plate allowing up to 40 patient samples per plate with results in ⁇ 3 hrs; 3) The use of primer mixtures can decrease the frequency of "no calls" often seen with other point mutation assays as a result of adjacent polymorphic mismatches; 4) This amplification-based technology is much more sensitive than conventional sequencing, and can be useful as both a primary screening tool and for post treatment evaluation; 5) This technology is currently used in public health lab settings and may be transferred to locations where current genotyping is cost-prohibited; and 6) Real-time PCR is a powerful tool that can garner simultaneous virologic measures (e.g., virus load and resistance load).
  • simultaneous virologic measures e.g., virus load and resistance load.
  • HIV-1 genomic RNA is extracted (Qiagen UltraSens RNA kit) from 200 ⁇ , plasma or serum and reconstituted in 50 ⁇ _, of buffer provided with the kit.
  • virus sequences are first amplified from 5 ⁇ , HIV-1 RNA by reverse transcriptase- polymerase chain reaction (RT-PCR) using the reverse primer of SEQ ID NO: 6, and forward primer of SEQ ID NO: 5.
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • PCR amplification conditions are 40 cycles of 95°C for 45 seconds, 50°C for 30 seconds, and 72°C for 2 minutes.
  • Real-time PCR-based mutation-specific assays for the 65R mutation in HIV-1 subtype C Mutation testing is performed in 96-well plates using 2 ⁇ . of 1 :20 dilutions of the RT-PCR products, except that samples with viral loads below 5000 copies/mL are not diluted.
  • the principle of the real-time PCR assay is to compare the differential amplifications of a mutation-specific PCR and a PCR that amplifies all viruses in the sample (total virus copy reaction) (FIG. 1).
  • the cycle number at which the fluorescence emission exceeds the background fluorescence threshold is the threshold cycle (CT) and is the unit of measure for comparing the differences in amplification signals (ACT) between the total copy and mutation-specific reactions (FIG. IB). All samples were tested in duplicate with the means of the total copy and mutation-specific CTs used for the determination of the ACT.
  • the mutation-specific primers of SEQ ID NOs: 1 18, 1 17 and 122 are designed to preferentially anneal with the targeted mutation nucleotide(s), thus having reduced affinity for wildtype sequences. To accommodate the various polymorphisms in large populations, degenerate nucleotides are placed at
  • primers complementary positions in the primers. Specificity is enhanced by creating designed mismatches at nucleotide(s) -2 to -4 positions from the primer 3'-end. Furthermore, to cover the spectrum of polymorphisms present, mixtures of multiple degenerate primers are often required. Mutation-specific primer mixtures are experimentally evaluated and the ratios that best balance differences in primer avidities and minimize cross-interference in primer annealing are selected. Each change is reevaluated against wildtype and mutant samples. The mutation specific forward primers are used in combination with a percent total primer concentration of each primer being SEQ ID NO: 118 (20%), SEQ ID NO: 117 (60%), and SEQ ID NO: 122 (20%).
  • Real-time PCRs are initiated with a hot-start incubation at 94°C for 11 minutes before proceeding to 45 cycles of melting at 94°C for 30 seconds, annealing at 50°C for 15 seconds and extension at 60°C for 30 seconds. All reactions are performed in a total volume of 25 or 50 ⁇ ⁇ in 96-well PCR plates using iCycler real-time PCR thermocyclers with optical units (Bio-Rad) and AmpliTaq Gold polymerase (2.5 U/reaction; Applied Biosystems). Final reagent concentrations are 320 nM for the forward and reverse primers, 160 nM probe(s), and 400 ⁇ dNTPs.
  • Relative limits of detection are compared in a simple laboratory setting using serial dilutions of cloned mutant template in a background of wildtype template.
  • the ACT that is equivalent to a 0.5 log greater reactivity than the wiidtype mean ACT on the dilution curve is used to compare assay sensitivities (FIG. 6). This approach yields a clinical detection limit of 2% and an absolute detection limits of 0.01%.
  • Example 3 Screening for HIV-1 Integrase Mutations.
  • Example 2 The methods of Example 2 are repeated using primers and probes for the specific detection of the HIV-1 integrase 148R mutation.
  • Reverse transcription is performed using primers of SEQ ID NOs: 124 and 125.
  • the DNA produced is then used in combination reaction amplifying two regions: 1) a common amplicon is produced using common primers SEQ ID NO: 26 and SEQ ID NO: 129; and 2) a mutation specific amplification reaction for the 148R mutation using mutation specific reverse primer SEQ ID NO: 138 and forward primer SEQ ID NO: 126.
  • the probes for the common amplicon and the 148R reaction are SEQ ID NOs: 140 (80%) and 141 (20%).
  • the absolute limits of detection obtained using these primers and probes is 0.4% (FIG. 7).
  • Patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are incorporated herein by reference to the same extent as if each individual application or publication was specifically and individually incorporated herein by reference

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Abstract

La présente invention concerne des compositions comprenant des amorces et des sondes, qui sont capables d'interagir avec les acides nucléiques décrits, tels que des acides nucléiques codant pour la transcriptase inverse, la protéase, ou l'intégrase du VIH. La présente invention concerne ainsi un oligonucléotide comprenant l'une quelconque des séquences nucléotidiques exposées dans SEQ ID NO : 1-89, 96-122, et 124-141. La présente invention concerne en outre les oligonucléotides constitués des nucléotides tels qu'exposés dans les SEQ ID NO : 1-89, 96- 122, et 124-141. Chacun des oligonucléotides décrits est une sonde ou une amorce. La présente invention concerne en outre des mélanges et des sondes destinés à être utilisés dans les réactions de RT-PCR et de PCR primaires décrites dans le présent document. La présente invention concerne des procédés de détection spécifique de plusieurs mutations dans le VIH, simultanément ou séquentiellement. Les mutations dans la transcriptase inverse, la protéase, ou l'intégrase du VIH peuvent être détectées à l'aide des procédés décrits dans le présent document.
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